Mimetic peptides derived from collagen type iv and their use for treating angiogenesis- and lymphangiogenesis- dependent diseases

ABSTRACT

Mimetic peptides having anti-angiogenic and anti-tumorigenic properties and methods of their use for treating cancer, ocular diseases, such as age-related macular degeneration, and other-angiogenesis-dependent diseases are disclosed. More particularly, active non-cysteine analogs (mimetics), which exhibit anti-angiogenic activity in endothelial cell proliferation, migration, adhesion, and tube formation assays, anti-migratory activity in human breast cancer cells in vitro, anti-angiogenic and anti-tumorigenic activity in vivo in breast cancer xenograft models, and age-related macular degeneration models are disclosed. The presently disclosed mimetic peptides also exhibit anti-lymphangiogenic and directly anti-tumorigenic properties.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.14/708,829 filed May 11, 2015, which is a Divisional of U.S. applicationSer. No. 13/992,998, filed on Aug. 7, 2013, which is a 35 U.S.C. §371National Stage Entry of International Application No. PCT/US2011/064475having an international filing date of Dec. 12, 2011, which claims thebenefit of U.S. Provisional Application Nos. 61/421,706, filed Dec. 10,2010; and 61/546,314, filed Oct. 12, 2011, each which are incorporatedherein by reference in their entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under R21 CA131931 andR01 CA138264 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains a sequence listing. It has been submittedelectronically via EFS-Web as an ASCII text file entitled“111232-00408_ST25.txt”. The sequence listing is 16,384 bytes in size,and was created on Jul. 20, 2015. It is hereby incorporated by referencein its entirety.

BACKGROUND

Cancer is a major public health problem in the United States and otherparts of the world. Currently, 1 in 4 deaths in the United States is dueto cancer. Angiogenesis plays a critical role in tumor growth andmetastasis in most types of cancer. In particular, its importance hasbeen demonstrated in breast cancer, the most commonly diagnosed femalemalignancy in the United States. Anti-angiogenic therapeutics, either asa monotherapy or in combination with other therapeutics, are promisingand are being intensely investigated in both preclinical and clinicalstudies. Anti-VEGF therapeutics showed early promise in clinical trials;however, although an anti-VEGF antibody bevacizumab (Genentech/Roche)was approved by the FDA for breast cancer in 2008 in combination withchemotherapy, in November 2011, the FDA revoked the breast cancerindication because it has not demonstrated an overall survival benefit.Accordingly, the development of anti-angiogenic therapies to treatbreast and other cancers, as well as, ocular proliferative diseases,such as age-related macular degeneration, is ongoing. Lymphangiogenesisalso plays an important role in cancer metastasis (Holopainen et al.,2011). To date no peptide drugs have been approved for the treatment ofcancer or other angiogenesis- and lymphangiogenesis-dependent diseases.

Peptides have been employed as therapeutics for multiple diseases andrecently have been investigated in clinical applications to targettumors either for imaging or therapy (Folkman, 2010; Senger et al.,1983; Leung et al., 1989; Carmeliet, 2005; Carmeliet and Jain, 2000;Carmeliet and Jain, 2011; Rosca et al., 2011). Mimetic peptides arepeptides that biologically mimic active determinants on biomolecules. Ingeneral, peptides are attractive tools as therapeutics due to theirspecific target binding, ability to penetrate cells and ease ofmodification giving flexibility for different applications. (Carmelietand Jain, 2000; Folkman, 2006) Some of the properties that make peptidesattractive candidates, however, also contribute to their disadvantages.Although peptides can interact specifically with cellular receptors,sometimes these interactions may be of low affinity. In addition, theuse of peptides as therapeutic agents is currently limited due to theirshort half-life and reduced bioavailability. Attempts to modify apeptide in order to increase its bioavailability include substitutionwith non-natural amino acids, pegylation of the peptide, and delivery ofthe peptide in a nano- or micro-particle.

SUMMARY

The presently disclosed subject matter provides peptide compositions,methods, and kits for treating a disease, disorder, or dysfunction thatis related to angiogenesis, lymphangiogenesis, vascular permeability ortumorigenesis. The presently disclosed peptide compositions and methods,in some aspects, inhibit angiogenesis, lymphangiogenesis, vascularpermeability or tumorigenesis which play a critical role in multiplediseases or disorders. Accordingly, in some aspects, the compositionsand methods of the presently disclosed subject matter allow theprevention or reduction of blood vessel, lymphatic vessel, or tumorformation involving a cell, tissue or organ.

More particularly, in some aspects, the presently disclosed subjectmatter provides compositions and methods of treating at least one cellwith an isolated peptide comprising the amino acid sequenceLRRFSTXPXXXXNINNVXNF (SEQ ID No:1), wherein X is any naturally occurringor non-naturally occurring amino acid.

In one aspect, the presently disclosed subject matter provides anisolated peptide comprising the amino acid sequence LRRFSTXPXXXXNINNVXNF(SEQ ID No:1), wherein X is any amino acid and wherein the peptide doesnot comprise LRRFSTMPFMFCNINNVCNF (SEQ ID No:19).

In another aspect, the presently disclosed subject matter provides anisolated peptide comprising the amino acid sequence LRRFSTXPXXXXNINNVXNF(SEQ ID No:1), wherein X at position 7 is M, A, or G; X at position 9 isF, A, Y, or G; X at position 10 is M, A, G, dA, or Nle; X at position 11is F, A, Y, G, or 4-ClPhe; X at position 12 and position 18 are Abu, G,S, A, V, T, I, L or AllyGly; and wherein the peptide does not compriseLRRFSTMPFMFCNINNVCNF (SEQ ID No:19).

In a further aspect, the presently disclosed subject matter provides anisolated peptide comprising at least one of the following amino acidsequences:

(SEQ ID No: 3) LRRFSTMPFMFAbuNINNVAbuNF; (SEQ ID No: 4)LRRFSTMPAMFAbuNINNVAbuNF; (SEQ ID No: 5) LRRFSTMPFAFAbuNINNVAbuNF;(SEQ ID No: 6) LRRFSTMPFMAAbuNINNVAbuNF; (SEQ ID No: 7)LRRFSTMPFNleFAbuNINNVAbuNF; (SEQ ID No. 8)LRRFSTMPFM4-ClPheAbuNINNVAbuNF; (SEQ ID No: 2) LRRFSTMPFMFGNINNVGNF;(SEQ ID No: 9) LRRFSTMPFMFSNINNVSNF; (SEQ ID No: 10)LRRFSTMPFMFANINNVANF; (SEQ ID No: 14) LRRFSTMPFMFVNINNVVNF;(SEQ ID No: 12) LRRFSTMPFMFTNINNVTNF; (SEQ ID No: 13)LRRFSTMPFMFAllyGlyNINNVAllyGlyNF; (SEQ ID No: 11) LRRFSTMPFMFININNVINF;(SEQ ID No. 15) LRRFSTMPFdAFININNVINF; (SEQ ID No. 16)LRRFSTAPFMFININNVINF; and (SEQ ID No. 17) LRRFSTAPFAFININNVINF.

In another aspect, the presently disclosed subject matter providescompositions and kits comprising a pharmaceutically acceptable carrierand an effective amount of at least one of the peptide sequencesdisclosed herein. The compositions and kits prevent or reduce bloodvessel, lymphatic vessel, or tumor formation involving a cell, tissue,or organ. The compositions and kits may also inhibit vascularpermeability involving a cell, tissue, or organ.

In a further aspect, the presently disclosed subject matter provides amethod for inhibiting angiogenesis, lymphangiogenesis, vascularpermeability and/or tumorigenesis involving a cell. The method comprisescontacting a cell with a presently disclosed isolated peptide in anamount sufficient to inhibit angiogenesis, lymphangiogenesis, vascularpermeability, and/or tumorigenesis of the cell or involving the cell.The contacting of the cell may result in an inhibition of adhesion,migration, proliferation, and/or tube formation of the cell.

Certain aspects of the presently disclosed subject matter provide forthe use of the isolated peptides in the treatment of a diseaseassociated with angiogenesis, lymphangiogenesis, vascular permeability,and/or tumorigenesis. The presently disclosed subject matter provides amethod of treating a subject suffering from a disease related toangiogenesis, lymphangiogenesis, vascular permeability, and/ortumorigenesis or preventing or delaying a subject from getting a diseaserelated to angiogenesis, lymphangiogenesis, vascular permeability,and/or tumorigenesis. The method comprises administering to the subjectan isolated peptide of the present invention in an amount sufficient totreat, delay, or prevent the disease in the subject. The disease may bea cancer or the disease may be related to ocular angiogenesis or otherangiogenesis-, tumorigenesis-, vascular permeability-, orlymphangiogenesis-dependent diseases.

The method of the presently disclosed subject matter can be practiced invivo as either a therapeutic method of treating a disease or disorderinvolving angiogenesis, lymphangiogenesis, vascular permeability, ortumorigenesis or as a prophylactic method to prevent angiogenesis,lymphangiogenesis, vascular permeability, or tumorigenesis. Likewise,the method can be practiced in vitro as a research tool to study theeffects of angiogenesis, lymphangiogenesis, vascular permeability ortumorigenesis. The method also can be practiced ex vivo for therapeuticor research purposes.

Certain aspects of the presently disclosed subject matter having beenstated hereinabove, which are addressed in whole or in part by thepresently disclosed subject matter, other aspects will become evident asthe description proceeds when taken in connection with the accompanyingExamples and Figures as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying Figures, which arenot necessarily drawn to scale, and wherein:

FIG. 1 shows the results from a proliferation assay for the full lengthpeptides with substituted Cysteines (SP2022-SP2027; SEQ ID Nos:9-14);

FIG. 2 illustrates an adhesion assay for the full length peptides withsubstituted Cysteines (SP2022-SP2027; SEQ ID Nos:9-14);

FIG. 3 demonstrates a migration assay for the full length peptides withsubstituted Cysteines (SP2022-SP2027; SEQ ID Nos:9-14);

FIGS. 4A and 4B show a wound healing assay with varied concentrations ofpeptide SP2012 (Panel A) and quantification of the assay (Panel B);

FIGS. 5A-5C illustrate an adhesion assay (Panel A), a migration assay at50 μM (Panel B), and a migration assay at 5 μM (Panel C) for the fulllength peptides with substituted Cysteines (SP2022-SP2027; SEQ IDNos:9-14);

FIG. 6A and FIG. 6B demonstrates a tube formation assay for the fulllength peptides with substituted Cysteines (SP2022-SP2027; SEQ IDNos:9-14);

FIG. 7 shows the results from a proliferation assay for the full lengthpeptides with Alanine and other substitutions (SP2015-SP2019; SEQ IDNos:4-8);

FIG. 8 illustrates an adhesion assay for the full length peptides withAlanine and other substitutions (SP2016-SP2019; SEQ ID Nos:5-8);

FIG. 9 demonstrates a migration assay for the full length peptides withAlanine and other substitutions (SP2015-SP2019; SEQ ID Nos:4-8);

FIG. 10 shows a tube formation assay for the peptide SP2015 (SEQ IDNo:4);

FIGS. 11A-11D illustrate the amino acid sequences of the C-terminaldeletion peptides (Panel A), an adhesion assay with these peptides(Panel B), a migration assay (Panel C), and inhibition of adhesion forSP2006 (SEQ ID No:18) (Panel D);

FIGS. 12A and 12B demonstrate the inhibitory activity of the C-terminaldeletion peptides in an adhesion assay (Panel A) and in a migrationassay (Panel B);

FIG. 13 shows a tube formation assay for the C-terminal deletionpeptides.

FIGS. 14A and 14B illustrate the inhibitory activity of the N-terminaldeletion peptides in an adhesion assay (Panel A) and in a migrationassay (Panel B);

FIGS. 15A-15D show the amino acid sequences of the peptides truncatedfrom both the N-terminal and C-terminal ends (Panel A), adhesion assayswith these peptides (Panel B), migration assays (Panel C), andproliferation assays (Panel D);

FIGS. 16A and 16B demonstrate adhesion assays (Panel A) and migrationassays (Panel B) with the peptides truncated from both the N-terminaland C-terminal ends;

FIG. 17 illustrates a tube formation assay with some of the N-terminaldeletion peptides and some of the peptides truncated on both ends;

FIGS. 18A and 18B show an adhesion assay (Panel A) and a migration assay(Panel B) with some of the full length peptides with other substitutions(SP2024, SP2034-SP2036; SEQ ID No:11, SEQ ID No:15-17);

FIGS. 19A and 19B demonstrate a proliferation assay (Panel A) and anadhesion assay (Panel B) with some of the full length peptides withother substitutions (SP2024, SP2034-SP2036; SEQ ID No:11, SEQ IDNo:15-17);

FIGS. 20A-20E illustrate a tube formation assay with many of thepeptides in the study;

FIGS. 21A-21E show the inhibitory activity of peptides SP2012 (SEQ IDNo:3) and SP2024 (SEQ ID No:11) in an adhesion assay (Panel A) andmigration assay (Panel C) on lymphatic endothelial cells, in an adhesionassay (Panel B) and migration assay (Panel D) on microvascular cells,and in a migration assay on breast cancer cells MDA-MB-231 (Panel E);

FIGS. 22A and 22B demonstrate the inhibitory activity of SP2024 (SEQ IDNo:11) on breast cancer cells MDA-MB-231 using an adhesion assay (PanelA) and a migration assay (Panel B);

FIGS. 23A and 23B illustrate the suppression of tumor growth by SP2000(SEQ ID No:19) and SP2012 (SEQ ID No:3) (Panel A) and the quantificationof microvascular density (Panel B);

FIGS. 24A-24C show the inhibition activity of SP2024 (SEQ ID No:11) ontumor growth (Panel A), relative vascular volume (Panel B), and tumorpermeability (Panel C);

FIG. 25 shows the inhibition of metastasis by SP2024 in an experimentalmetastasis model of breast cancer;

FIGS. 26A and 26B demonstrate the results from the injection of SP2024(SEQ ID No:11) into a mouse eye by assaying the area of choroidalneovascularization (Panel A) and the area of retinal neovascularization(Panel B); and

FIGS. 27A and 27B illustrate the results from injection of SP2034,SP2035, and SP2036 (SEQ ID Nos:15-17) into a mouse eye by assayinglaser-induced choroidal neovascularization (Panel A) and the area ofretinal neovascularization in the Rho/VEGF model (Panel B);

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fullyhereinafter with reference to the accompanying Figures, in which some,but not all embodiments of the presently disclosed subject matter areshown. Like numbers refer to like elements throughout. The presentlydisclosed subject matter may be embodied in many different forms andshould not be construed as limited to the embodiments set forth herein;rather, these embodiments are provided so that this disclosure willsatisfy applicable legal requirements. Indeed, many modifications andother embodiments of the presently disclosed subject matter set forthherein will come to mind to one skilled in the art to which thepresently disclosed subject matter pertains having the benefit of theteachings presented in the foregoing descriptions and the associatedFigures. Therefore, it is to be understood that the presently disclosedsubject matter is not to be limited to the specific embodimentsdisclosed and that modifications and other embodiments are intended tobe included within the scope of the appended claims.

I. Mimetic Peptides Derived from Collagen Type IV

Peptides generally offer many advantages over other types of therapiesfor certain diseases in that they are non-immunogenic, less toxicbecause they bind to their targets with high specificity, and areinexpensive to produce (e.g., International Publication WO 2008/085828and International Publication WO 2007/033215, each which is incorporatedherein by reference in its entirety). The presently disclosed peptidesexhibit anti-angiogenic, anti-vascular permeability, anti-tumorigenesisand/or anti-lymphangiogenic properties, which could lead to an increasein overall survival in certain diseases. For example, the peptides maybenefit cancer patients and may help treat ocular proliferativediseases, such as age-related macular degeneration. In general, thepresently disclosed peptides are characterized by having the motifcomprising the amino acid sequence LRRFSTXPXXXXNINNVXNF (SEQ ID No:1).

A. Representative Embodiments

In one embodiment, the peptide comprises the motif LRRFSTXPXXXXNINNVXNF(SEQ ID No: 1), X is any amino acid and the peptide does not compriseLRRFSTMPFMFCNINNVCNF (SEQ ID No:19), a previously disclosed peptide(Popel A. S. and Karagiannis E. D. “Peptide Inhibitors or Modifiers ofAngiogenesis and Uses Thereof,” PCT/WO/2008/085828; Rosca et al., 2011).X may be a natural or non-natural amino acid.

The nomenclature used herein is three-fold. First, all peptides beginwith the letters SP; the peptide series is denoted by the first numberand the last three numbers denote the particular peptide in the series.Thus, the presently disclosed series of peptides is labeled SP2XXX, withpentastatin 1, the parent, being labeled SP2000 (SEQ ID No:19).

The presently disclosed peptide series labeled SP2XXX were tested inangiogenesis and lymphangiogenesis assays in vitro. More particularly,proliferation, adhesion, migration, and tube formation assays wereperformed on endothelial, e.g., Human Umbilical Vein Endothelial Cells(HUVECs), and other cells in vitro. Selected peptides were also testedin vivo in tumor xenograft and ocular assays. Representative embodimentsof the peptide series are shown in Table 1 and are discussed in moredetail below.

TABLE 1 Representative Presently Disclosed Peptides SEQ ID No: 3LRRFSTMPF M F Abu NINNV Abu  SP2012 NF SEQ ID No: 4LRRFSTMPA M F Abu NINNV Abu  SP2015 NF SEQ ID No: 5LRRFSTMPF A F Abu NINNV Abu  SP2016 NF SEQ ID No: 6LRRFSTMPF M A Abu NINNV Abu  SP2017 NF SEQ ID No: 7LRRFSTMPF Nle F Abu NINNV Abu SP2018 NF SEQ ID No: 8LRRFSTMPF M 4-ClPhe Abu NINNV SP2019 Abu NF SEQ ID No: 2LRRFSTMPF M F G NINNV G NF SP2002 SEQ ID No: 9LRRFSTMPF M F S NINNV S NF SP2022 SEQ ID No: 10LRRFSTMPF M F A NINNV A NF SP2023 SEQ ID No: 14LRRFSTMPF M F V NINNV V NF SP2027 SEQ ID No: 12LRRFSTMPF M F T NINNV T NF SP2025 SEQ ID No: 13 LRRFSTMPF M F AllyGly SP2026 NINNVAllyGly NF SEQ ID No: 11 LRRFSTMPF M F I NINNV I NF SP2024SEQ ID No: 15 LRRFSTMPF dA F I NINNV I NF SP2034 SEQ ID No: 16LRRFSTAPF M F I NINNV I NF SP2035 SEQ ID No: 17LRRFSTAPF A F I NINNV I NF SP2036

For the presently disclosed subject matter, the peptides have several Xresidues that may be any amino acid, whether natural or non-natural (X7,X9, X10, X11, X12, and X18). By natural amino acids, it is meant thoseamino acids that occur in nature. By non-natural amino acids, it ismeant amino acids that do not occur in nature but that can beincorporated into a polypeptide chain. Non-natural amino acids include,but are not limited to 2-aminobutyric acid (Abu), norleucine (Nle),4-chloro phenylalanine (4-ClPhe), allylglycine (AllyGly) and othernon-natural amino acids such as those detailed in Ma (2003). Amino acidanalogs that are known in the art may be employed in the presentinvention.

A “peptide” or “protein” comprises a string of at least three aminoacids linked together by peptide bonds. The terms “protein” and“peptide” may be used interchangeably. Peptide may refer to anindividual peptide or a collection of peptides. Also, one or more of theamino acids in a presently disclosed peptide may be modified, forexample, by the addition of a chemical entity such as a carbohydrategroup, a phosphate group, a farnesyl group, an isofarnesyl group, afatty acid group, a linker for conjugation, functionalization, or othermodification, and the like. In some embodiments, the modifications ofthe peptide lead to a more stable peptide (e.g., greater half-life invivo). These modifications may include cyclization of the peptide, theincorporation of D-amino acids, and the like. None of the modificationsshould substantially interfere with the desired biological activity ofthe peptide.

By “Collagen IV derived peptide” it is meant a peptide comprising aC-N-X(3)-V-C or P-F-X(2)-C or LX(2)FX(3)PFX(2)CNX(4)CNX collagen motif.If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 ormore amino acids that flank the carboxy or amino terminus of the motifin the naturally occurring amino acid sequence. Type IV collagen derivedpeptides include, for example, pentastatin-1, tumstatin, and targetingRGD. By “alteration” is meant a change in the sequence or in amodification (e.g., a post-translational modification) of a gene orpolypeptide relative to an endogeneous wild-type reference sequence.

By an “isolated peptide” is meant a peptide of the invention that hasbeen separated from components that naturally accompany it. Typically,the peptide is isolated when it is at least 60%, by weight, free fromthe proteins and naturally-occurring organic molecules with which it isnaturally associated. Preferably, the preparation is at least 75%, morepreferably at least 90%, and most preferably at least 99%, by weight, apresently disclosed peptide of the invention. An isolated peptide may beobtained, for example, by extraction from a natural source, byexpression of a recombinant nucleic acid encoding such a peptide; or bychemically synthesizing the protein. Purity can be measured by anyappropriate method, for example, column chromatography, polyacrylamidegel electrophoresis, or by HPLC analysis.

By “substantially identical” is meant a peptide, a polypeptide ornucleic acid molecule exhibiting at least 50% identity to a referenceamino acid sequence (for example, any one of the amino acid sequencesdescribed herein) or nucleic acid sequence. Preferably, such a sequenceis at least 60%, more preferably 80% or 85%, and even more preferably90%, 95% or even 99% identical at the amino acid level or nucleic acidto the sequence used for comparison.

Further, the presently disclosed peptides can be modified to make themless susceptible to proteolysis. For example, they can be truncated tothe minimal potent sequence. Such truncation is important to limitbinding to other receptors that would dilute the effectiveconcentration, as well as lead to unexpected side effects. Suchtruncation also opens up the possibility to create a single multimodalpeptide out of multiple short peptides, each of which targetsangiogenesis, lymphangiogenesis and tumorigenesis by a differentmechanism. Multimodal treatment is very important to reduce theincidence of drug resistance, because it is less likely that the tumorwill be able to mount a successful resistance when attackedsimultaneously from multiple fronts.

In addition, the presently disclosed peptides with different sequencescan be used together in one composition or method. There may becompositions or methods where multiple types of the presently disclosedpeptides allow better prevention or reduction of angiogenesis, vascularpermeability, tumorigenesis or lymphangiogenesis. Therefore, instead ofa composition with a single multimodal peptide, a composition may becomprised of multiple types of isolated peptides that are not covalentlybound together.

Further, the presently disclosed peptides are all tri-fluoro acetate(TFA) salts. For use in humans, however, the TFA salts can be modifiedto acetate salts or other pharmaceutically acceptable salts.

The term “pharmaceutically acceptable salts” is meant to include saltsof active compounds which are prepared with relatively nontoxic acids orbases, depending on the particular substituent moieties found on thecompounds described herein. When compounds of the present disclosurecontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentdisclosure contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike {see, for example, Berge et al., “Pharmaceutical Salts”, Journal ofPharmaceutical Science, 1977, 66, 1-19). Certain specific compounds ofthe present disclosure contain both basic and acidic functionalitiesthat allow the compounds to be converted into either base or acidaddition salts.

Further, one or more of the presently disclosed peptides can beincorporated into nano or microparticles, such as those disclosed inU.S. patent application Ser. No. 13/272,042, to Green et al. for“Peptide/Particle Delivery Systems,” filed Oct. 12, 2011, andInternational PCT Patent Application Publication No. WO/2010/132879 toGreen et al. for “Multicomponent Degradable Cationic Polymers,” filedMay 17, 2010, both of which are commonly owned and incorporated hereinby reference in their entirety.

In addition, it is possible to increase the half-lives of the peptidesby conjugating the peptides to certain compounds. For example, it ispossible to conjugate the peptides to catalytic antibodies to increasetheir half-lives.

In an embodiment, the presently disclosed subject matter provides anisolated peptide comprising the amino acid sequence LRRFSTXPXXXXNINNVXNF(SEQ ID No:1), wherein X at position 7 is M, A, or G; X at position 9 isF, A, Y, or G; X at position 10 is M, A, G, dA, or Nle; X at position 11is F, A, Y, G, or 4-ClPhe; X at position 12 and position 18 are Abu, G,S, A, V, T, I, L or AllyGly; and wherein the peptide does not compriseLRRFSTMPFMFCNINNVCNF (SEQ ID No:19).

In a further embodiment, the presently disclosed subject matter providesan isolated peptide comprising at least one of the following amino acidsequences:

(SEQ ID No: 3) LRRFSTMPFMFAbuNINNVAbuNF; (SEQ ID No: 4)LRRFSTMPAMFAbuNINNVAbuNF; (SEQ ID No: 5) LRRFSTMPFAFAbuNINNVAbuNF;(SEQ ID No: 6) LRRFSTMPFMAAbuNINNVAbuNF; (SEQ ID No: 7)LRRFSTMPFNleFAbuNINNVAbuNF; (SEQ ID No: 8)LRRFSTMPFM4-ClPheAbuNINNVAbuNF; (SEQ ID No: 2) LRRFSTMPFMFGNINNVGNF;(SEQ ID No: 9) LRRFSTMPFMFSNINNVSNF; (SEQ ID No: 10)LRRFSTMPFMFANINNVANF; (SEQ ID No: 14) LRRFSTMPFMFVNINNVVNF;(SEQ ID No: 12) LRRFSTMPFMFTNINNVTNF; (SEQ ID No: 13)LRRFSTMPFMFAllyGlyNINNVAllyGlyNF; (SEQ ID No: 11) LRRFSTMPFMFININNVINF;(SEQ ID No: 15) LRRFSTMPFdAFININNVINF; (SEQ ID No: 16)LRRFSTAPFMFININNVINF; and (SEQ ID No: 17) LRRFSTAPFAFININNVINF.

In another embodiment, the presently disclosed subject matter providescompositions and kits comprising a pharmaceutically acceptable carrierand an effective amount of at least one of the peptide sequencesdisclosed herein. The amount of peptide can vary widely, but generallythe amount is sufficient to perform at least one method of theinvention.

As used herein, “pharmaceutically acceptable carrier” is intended toinclude, but is not limited to, water, saline, dextrose solutions, humanserum albumin, liposomes, hydrogels, microparticles and nanoparticles.The use of such media and agents for pharmaceutically activecompositions is well known in the art, and thus further examples andmethods of incorporating each into compositions at effective levels neednot be discussed here. Such compositions also can include coatings,antibacterial and/or fungal agents, and any other ingredient that isbiologically tolerable.

In general, the “effective amount” of an active agent or drug deliverydevice refers to the amount necessary to elicit the desired biologicalresponse. As will be appreciated by those of ordinary skill in this art,the effective amount of an agent or device may vary depending on suchfactors as the desired biological endpoint, the agent to be delivered,the composition of the encapsulating matrix, the target tissue, and thelike.

In general, a presently disclosed kit contains some or all of thecomponents, reagents, supplies, and the like to practice a methodaccording to the presently disclosed subject matter. In a kit comprisingan isolated peptide according to the presently disclosed subject matter,the kit typically comprises an effective amount of peptide to prevent,delay, reduce, or treat a disease related to angiogenesis and/orlymphangiogenesis. In one embodiment, a kit comprises at least onecontainer (e.g. a vial, tube, or ampoule) comprising an isolated peptideof the presently disclosed subject matter. Typically, the isolatedpeptide or peptides will be supplied in one or more container, eachcontainer containing an effective amount of isolated peptide to allow achange in angiogenesis and/or lymphangiogenesis to occur.

B. General Terms

For clarity, other general terms are described below. Sequence identityis typically measured using sequence analysis software (for example,Sequence Analysis Software Package of the Genetics Computer Group,University of Wisconsin Biotechnology Center, 1710 University Avenue,Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs).Such software matches identical or similar sequences by assigningdegrees of homology to various substitutions, deletions, and/or othermodifications. Conservative substitutions typically includesubstitutions within the following groups: glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid, asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine. In an exemplary approach to determining the degree ofidentity, a BLAST program may be used, with a probability score betweene⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

“Sequence identity” or “identity” in the context of two nucleic acid orpolypeptide sequences includes reference to the residues in the twosequences which are the same when aligned for maximum correspondenceover a specified comparison window, and can take into considerationadditions, deletions and substitutions. When percentage of sequenceidentity is used in reference to proteins it is recognized that residuepositions which are not identical often differ by conservative aminoacid substitutions, where amino acid residues are substituted for otheramino acid residues with similar chemical properties (for example,charge or hydrophobicity) and therefore do not deleteriously change thefunctional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences which differ by such conservative substitutionsare said to have sequence similarity. Approaches for making thisadjustment are well-known to those of skill in the art. Typically thisinvolves scoring a conservative substitution as a partial rather than afull mismatch, thereby increasing the percentage sequence identity.Thus, for example, where an identical amino acid is given a score of 1and a non-conservative substitution is given a score of zero, aconservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, for example,according to the algorithm of Meyers and Miller, Computer Applic. Biol.Sci., 4: 11-17, 1988, for example, as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif., USA).

“Percentage of sequence identity” means the value determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions, substitutions, or deletions (i.e., gaps)as compared to the reference sequence (which does not compriseadditions, substitutions, or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity.

The term “substantial identity” or “homologous” in their variousgrammatical forms in the context of polynucleotides means that apolynucleotide comprises a sequence that has a desired identity, forexample, at least 60% identity, preferably at least 70% sequenceidentity, more preferably at least 80%, still more preferably at least90% and even more preferably at least 95%, compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning and thelike. Substantial identity of amino acid sequences for these purposesnormally means sequence identity of at least 60%, more preferably atleast 70%, 80%, 85%, 90%, and even more preferably at least 95%.

A “reference sequence” is a defined sequence used as a basis forsequence comparison. A reference sequence may be a subset of or theentirety of a specified sequence; for example, a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence. For polypeptides, the length of the reference polypeptidesequence will generally be at least about 5, 10, or 15 amino acids,preferably at least about 20 amino acids, more preferably at least about25 amino acids, and even more preferably about 35 amino acids, about 50amino acids, about 100 amino acids, or about 150 amino acids.

II. Methods of Treating Angiogenesis- and Lymphangiogenesis-DependentDiseases A. Representative Embodiments

In some embodiments, the presently disclosed peptides exhibitanti-angiogenic anti-lymphangiogenic, anti-tumorigenic, and/oranti-vascular permeable properties. Angiogenesis refers to the growth ofnew blood vessels originating from existing blood vessels.Lymphangiogenesis refers to the formation of lymphatic vessels frompre-existing lymphatic vessels, in a method believed to be similar toblood vessel development or angiogenesis. Tumorigenesis refers to theformation of a tumor. Vascular permeability refers to the property ofblood capillary walls that allows for the selective exchange ofsubstances.

In some embodiments, the presently disclosed subject matter provides amethod of inhibiting angiogenesis and/or lymphangiogenesis involving acell. The method comprises contacting a cell with a presently disclosedisolated peptide in an amount sufficient to inhibit angiogenesis and/orlymphangiogenesis of the cell. The contacting of the cell may result inan inhibition of adhesion, migration, proliferation, and/or tubeformation involving the cell. In a particular embodiment, the cell is anendothelial cell.

The method of the presently disclosed subject matter can be practiced invivo as either a therapeutic method of treating a disease or disorderinvolving angiogenesis or lymphangiogenesis or as a prophylactic methodto prevent angiogenesis or lymphangiogenesis. Likewise, the method canbe practiced in vitro as a research tool to study the effects ofangiogenesis or lymphangiogenesis on a cell. The method also can bepracticed ex vivo for therapeutic or research purposes.

“Contacting” means any action that results in at least one isolatedpeptide of the presently disclosed subject matter physically contactingat least one cell. It thus may comprise exposing the cell(s) to theisolated peptide in an amount sufficient to result in contact of atleast one isolated peptide with at least one cell. The method can bepracticed in vitro or ex vivo by introducing, and preferably mixing, theisolated peptide and cells in a controlled environment, such as aculture dish or tube. The method can be practiced in vivo, in which casecontacting means exposing at least one cell in a subject to at least oneisolated peptide of the presently disclosed subject matter, such asadministering the isolated peptide to a subject via any suitable route.According to the presently disclosed subject matter, contacting maycomprise introducing, exposing, and the like, the isolated peptide at asite distant to the cells to be contacted, and allowing the bodilyfunctions of the subject, or natural (e.g., diffusion) or man-induced(e.g., swirling) movements of fluids to result in contact of theisolated peptide and cell(s).

In some embodiments, the presently disclosed subject matter provides amethod for treating a subject suffering from a disease related toangiogenesis, lymphangiogenesis, tumorigenesis, and/or vascularpermeability or to prevent or delay a subject from developing a diseaserelated to angiogenesis, lymphangiogenesis, tumorigenesis, and/orvascular permeability. The method comprises administering to the subjecta presently disclosed isolated peptide in an amount sufficient to treat,delay, or prevent the disease in the subject. Representative diseasesinclude those diseases that are angiogenesis-, lymphangiogenesis-,tumorigenesis-, and or vascular permeability-dependent. Accordingly, insome embodiments, the disease may be a cancer, such as cancer of thebreast, lung, glioblastoma, renal cell, hepatic cell, head, neck, or anyother cancer that relies on angiogenesis or lymphangiogenesis to occuror spread. In one embodiment, the method treats a primary tumor. Inanother embodiment, the method treats an established metastasized tumor.The method may inhibit angiogenesis and/or lymphangiogenesis in orsurrounding a tumor. The method also may inhibit dissemination of tumorcells through the blood and/or lymphatic vasculature. Further, themethod may inhibit the establishment of metastasis or inhibit furthermetastasis of the cancer.

In other embodiments, the disease may be related to ocular angiogenesis,such as age-related macular degeneration, macular edema, neovascularglaucoma, proliferative diabetic retinopathy, or retinopathy ofprematurity. In vivo experiments described herein below illustrate theeffect of selected isolated peptides of the presently disclosed subjectmatter in a mouse model.

In certain embodiments, the presently disclosed subject matter providesfor the use of the isolated peptides in the treatment of a diseaseassociated with angiogenesis, lymphangiogenesis, tumorigenesis, and/orvascular permeability. The use is in particular for in vivo therapeuticor prophylactic methods of inhibiting angiogenesis, lymphangiogenesis,tumorigenesis, and/or vascular permeability. Certain embodiments providefor the use of the isolated peptides in the preparation of compositionsfor medical use, such as pharmaceutical or therapeutic compositions. Ingeneral, use of the isolated peptides is in combining them with othersubstances to make medicinal compositions.

The peptides according to the disclosure are effective over a widedosage range. For example, in the treatment of adult humans, dosagesfrom 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, andfrom 5 to 40 mg per day are examples of dosages that may be used. Anon-limiting dosage is 10 to 30 mg per day. The exact dosage will dependupon the route of administration, the form in which the compound isadministered, the subject to be treated, the body weight of the subjectto be treated, and the preference and experience of the attendingphysician.

B. General Terms

By “disease” is meant any condition, dysfunction or disorder thatdamages or interferes with the normal function of a cell, tissue, ororgan.

A “cancer” in an animal refers to the presence of cells possessingcharacteristics typical of cancer-causing cells, for example,uncontrolled proliferation, loss of specialized functions, immortality,significant metastatic potential, significant increase in anti-apoptoticactivity, rapid growth and proliferation rate, and certaincharacteristic morphology and cellular markers. In some circumstances,cancer cells will be in the form of a tumor; such cells may existlocally within an animal, or circulate in the blood stream asindependent cells, for example, leukemic cells.

By “blood vessel formation” is meant the dynamic process that includesone or more steps of blood vessel development and/or maturation, such asangiogenesis, vasculogenesis, formation of an immature blood vesselnetwork, blood vessel remodeling, blood vessel stabilization, bloodvessel maturation, blood vessel differentiation, or establishment of afunctional blood vessel network.

By “vasculogenesis” is meant the development of new blood vesselsoriginating from stem cells, angioblasts, or other precursor cells.

By “blood vessel stability” is meant the maintenance of a blood vesselnetwork.

By “neoplasia” is meant a disease that is caused by or results ininappropriately high levels of cell division, inappropriately low levelsof apoptosis, or both. Solid tumors, hematological disorders, andcancers are examples of neoplasias.

A “tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all precancerous andcancerous cells and tissues.

As used herein, the terms “treat,” “treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disorder or condition in a subject, who does not have,but is at risk of or susceptible to developing a disorder or condition.

By “reduce” is meant a decrease in a parameter (e.g., blood vesselformation) as detected by standard art known methods, such as thosedescribed herein. As used herein, reduce includes a 10% change,preferably a 25% change, more preferably a 40% change, and even morepreferably a 50% or greater change.

The subject treated by the presently disclosed methods in their manyembodiments is desirably a human subject, although it is to beunderstood that the methods described herein are effective with respectto all vertebrate species, which are intended to be included in the term“subject.” Accordingly, a “subject” can include a human subject formedical purposes, such as for the treatment of an existing condition ordisease or the prophylactic treatment for preventing the onset of acondition or disease, or an animal subject for medical, veterinarypurposes, or developmental purposes. Suitable animal subjects includemammals including, but not limited to, primates, e.g., humans, monkeys,apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines,e.g., sheep and the like; caprines, e.g., goats and the like; porcines,e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras,and the like; felines, including wild and domestic cats; canines,including dogs; lagomorphs, including rabbits, hares, and the like; androdents, including mice, rats, and the like. An animal may be atransgenic animal. In some embodiments, the subject is a humanincluding, but not limited to, fetal, neonatal, infant, juvenile, andadult subjects. Further, a “subject” can include a patient afflictedwith or suspected of being afflicted with a condition or disease. Thus,the terms “subject” and “patient” are used interchangeably herein.

In therapeutic and/or diagnostic applications, the compounds of thedisclosure can be formulated for a variety of modes of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remington: The Science andPractice of Pharmacy (20^(th) ed.) Lippincott, Williams & Wilkins(2000). To aid in bioavailability, the compounds of the disclosure maybe delivered in a nano- or micro-particles.

Pharmaceutically acceptable salts are generally well known to those ofordinary skill in the art, and may include, by way of example but notlimitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate,bitartrate, bromide, calcium edetate, carnsylate, carbonate, citrate,edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate,napsylate, nitrate, pamoate (embonate), pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Otherpharmaceutically acceptable salts may be found in, for example,Remington: The Science and Practice of Pharmacy (20^(th) ed.)Lippincott, Williams & Wilkins (2000). Pharmaceutically acceptable saltsinclude, for example, acetate, benzoate, bromide, carbonate, citrate,gluconate, hydrobromide, hydrochloride, maleate, mesylate, napsylate,pamoate (embonate), phosphate, salicylate, succinate, sulfate, ortartrate.

Depending on the specific conditions being treated, such agents may beformulated into liquid or solid dosage forms and administeredsystemically or locally. The agents may be delivered, for example, in atimed- or sustained-low release form as is known to those skilled in theart. Techniques for formulation and administration may be found inRemington: The Science and Practice of Pharmacy (20^(th) ed.)Lippincott, Williams & Wilkins (2000). Suitable routes may include oral,buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal,transmucosal, nasal or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intra-articullar, intra-sternal, intra-synovial, intra-hepatic,intralesional, intracranial, intraperitoneal, intranasal, or intraocularinjections or other modes of delivery.

For injection, the agents of the disclosure may be formulated anddiluted in aqueous solutions, such as in physiologically compatiblebuffers such as Hank's solution, Ringer's solution, or physiologicalsaline buffer. For such transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

Use of pharmaceutically acceptable inert carriers to formulate thecompounds herein disclosed for the practice of the disclosure intodosages suitable for systemic administration is within the scope of thedisclosure. With proper choice of carrier and suitable manufacturingpractice, the compositions of the present disclosure, in particular,those formulated as solutions, may be administered parenterally, such asby intravenous injection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe disclosure to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya subject (e.g., patient) to be treated.

For nasal or inhalation delivery, the agents of the disclosure also maybe formulated by methods known to those of skill in the art, and mayinclude, for example, but not limited to, examples of solubilizing,diluting, or dispersing substances such as, saline, preservatives, suchas benzyl alcohol, absorption promoters, and fluorocarbons.

Pharmaceutical compositions suitable for use in the present disclosureinclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipients, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations, for example, maize starch, wheat starch, rice starch,potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC),and/or polyvinylpyrrolidone (PVP: povidone). If desired, disintegratingagents may be added, such as the cross-linked polyvinylpyrrolidone,agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol (PEG), and/or titanium dioxide, lacquer solutions, and suitableorganic solvents or solvent mixtures. Dye-stuffs or pigments may beadded to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin, and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols (PEGs). In addition, stabilizers may be added.

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this presently described subject matter belongs.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a subject” includes aplurality of subjects, unless the context clearly is to the contrary(e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing amounts, sizes, dimensions,proportions, shapes, formulations, parameters, percentages, parameters,quantities, characteristics, and other numerical values used in thespecification and claims, are to be understood as being modified in allinstances by the term “about” even though the term “about” may notexpressly appear with the value, amount or range. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are not and need not beexact, but may be approximate and/or larger or smaller as desired,reflecting tolerances, conversion factors, rounding off, measurementerror and the like, and other factors known to those of skill in the artdepending on the desired properties sought to be obtained by thepresently disclosed subject matter. For example, the term “about,” whenreferring to a value can be meant to encompass variations of, in someembodiments, ±100% in some embodiments ±50%, in some embodiments ±20%,in some embodiments ±10%, in some embodiments ±5%, in some embodiments±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from thespecified amount, as such variations are appropriate to perform thedisclosed methods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or morenumbers or numerical ranges, should be understood to refer to all suchnumbers, including all numbers in a range and modifies that range byextending the boundaries above and below the numerical values set forth.The recitation of numerical ranges by endpoints includes all numbers,e.g., whole integers, including fractions thereof, subsumed within thatrange (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5,as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like)and any range within that range.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The following Examples are offered by way ofillustration and not by way of limitation.

Example 1 Cysteine Substitutions in the Full Peptide

SP2000 (SEQ ID No:19), which has the sequence LRRFSTMPFMFCNINNVCNF, wasthe original peptide identified by a previously disclosed Bioinformaticsapproach (Karagiannis and Popel, 2008). In this Example, the cysteinesin SP2000 (SEQ ID No:19) were substituted to determine if the twocysteines are essential for activity (Table 2).

For the growth of cells in cell culture, human umbilical veinendothelial cells (HUVEC), microvascular endothelial cells (MEC), andlymphatic endothelial cells (LEC) were purchased from Lonza andmaintained according to the manufacturer's recommendation usingEndothelial Basal Media (EBM-2) supplemented with the Bullet Kit (EGM-2,Lonza). The MEC and LEC were propagated in Microvascular EndothelialCell Growth Medium-2 (EGM-2MV, Lonza). Breast cancer cells, MDA-MB-231were supplied by Dr. Zaver Bhujwalla (JHMI, Radiology and Oncology). Thecells were propagated in RPMI-1640 medium (Gibco, Carlsbad, Calif.)supplemented with 10% FBS and antibiotics (1% penicillin/streptomycin).Cells were maintained under standard conditions of 37° C. and 5% CO₂ andthe passage numbers of all used cells were between 2 and 7.

Peptides were synthesized using solid-phase synthesis and were suppliedas TFA salts with an amidated C-terminus by two vendors (New EnglandPeptide, Gardner, Mass. and American Peptide Company, Sunnyvale,Calif.). The purity of the peptides was >95% and the suppliers providedproduct characterization (MALDI-TOF and HPLC traces) as proof of MW andpurity accuracy. Peptides were solubilized in 5% DMSO and water due totheir hydrophobic profile. The pH of solubilized peptides was checkedand found to be around pH 7. For all experiments the DMSO % wasmaintained at non-toxic threshold (determined by toxicity curves of DMSOon cells) with a final DMSO percentage (<0.2%) which was used as controlin all experiments.

Several assays were used to test the peptides discussed in the Examplesherein. A colorimetric based proliferation assay using WST-1 (Roche,11644807001) proliferation reagent was carried out using HUVEC cells.2000 cells/well were plated in 96-well plates and allowed to adhereovernight. On the following day, the media was exchanged with fullysupplemented media containing peptides or equivalent DMSO vehicle forthe controls. Three days later the media containing the peptides wasreplaced with serum-free EBM-2 media containing WST-1 reagent and theplates were incubated for four hours as per the manufacturer'srecommendations. Changes in color, due to the formazan dye resulted fromthe cleavage of the tetrazolium salt WST-1 by the mitochondrialsuccinate-tetrazolium reductase, were read on a Victor V fluorescenceplate reader (Perkin Elmer, Mass.) by measuring the absorbance at 450nm. Dose response curves of percent live cells (in comparison tountreated cells but incubated in complete media with 0.2% DMSO) werecreated. Assays were performed in at least two independent replicatesand each replicate was performed using three experimental triplicates.

The inhibitory potential of the peptides was measured using a real timemigration assay system based on electrical impedance (RT-CIM, ACEABiosciences, CA). CIM 16 well plates (Roche, 05665817001) are composedof a top and bottom chamber separated by a microporous (8 μm)polycarbonate membrane. The membrane was coated with fibronectin (20μg/mL), and 45,000 cells/well in serum free media with or withoutpeptides were added to the top compartment. Media with chemoattractant(i.e., fully supplemented EBM-2) was added to the bottom compartment ofthe chamber and the plate was incubated at 37° C. for 20 hours. Thesensors integrated on the bottom side of the membrane monitor andcontinuously record changes in impedance as the cells move through themembrane. The RT-CIM technology allows for easy quantification of cellmigration by monitoring the cell index derived from the measuredimpedances. Assays were performed in at least two independent replicatesand each replicate was performed using two experimental duplicates.

Breast cancer cells MDA-MB-231 are not suitable for the RT-CIM typeexperiments due to their thin elongated phenotype, thus the inhibitionof migration was investigated using a wound healing type assay. Thisassay was performed using the Oris Pro Migration assay (PlatypusTechnologies, CMA 1.101). Briefly, 20,000-25,000 cells/well in fullmedia were added to the 96 well plate containing stoppers to blockmigration of cells to the center region of the wells. Cells were allowedto adhere for 4 hours, after which the stoppers were removed. Cells werewashed one time with PBS and fully supplemented media, with or withoutcompound, was added to the wells. After 18 hours, cells were stainedwith calcein AM (0.5 μg/mL) (Invitrogen, CA) and the cells that migratedto the center of the well were quantified by fluorescence intensitymeasured using a Victor V plate reader (Perkin Elmer) and also imagedusing a Leica or Nikon microscope. The detection of the cells thatmigrated into the previously restricted region was possible due to theaddition of a detection mask at the bottom of the plate, whichobstructed from measurement cells that did not migrate.

Similar to the migration assays, the inhibition activity of the peptidein cellular adhesion was assessed using the RT-CIM technology. In thiscase, 25,000 cells/well were plated in 16 wells E-plates (Roche, BaselSwitzerland) in the presence or absence of the peptide. The adhesion wasmonitored over time (3 hours) by measuring changes in the electricalimpedance, which is a direct measure of the cells adhering on theelectrodes. For the most active peptide, IC₅₀ values were calculatedfrom dose response curves. Assays were performed in at least twoindependent replicates and each replicate was performed using twoexperimental duplicates.

The compounds also were tested for their ability to inhibit tubeformation, a process critical in angiogenesis. Endothelial cellsspontaneously form a network of tubes when plated on extracts ofextracellular matrix. This in vitro assay combines aspects of adhesionand migration and it is routinely used in angiogenesis research(Oliveira-Ferrer et al., 2008). The ability to inhibit tube formation isa comprehensive assessment of the anti-angiogenic potential. Theprotocol was described by Arnaoutova et al. (2009) and it consists ofplating HUVEC on top of basement membrane extract and after incubationat 37° C. the cells naturally rearrange themselves in a network oftubes. Thus, 50 μL/well of Matrigel (BD Biosciences, San Jose, Calif.)was plated in a cold 96 well plate and incubated at 37° C. for 30 minfor polymerization. 15,000 cells/well were added to the top of the geland incubated in complete media in the presence or absence of peptidefor 19 hours. Images were captured using the CCD Sensicam mounted on aNikon microscope (Eclipse T-100). Assays were performed in at least twoindependent replicates and each replicate was performed using threeexperimental replicates and one image of a randomly chosen field wasacquired per well.

For primary tumor inhibition studies, 1-2×10⁶ tumor cells were injectedinto 4-6 wk old female SCID mice. After the tumors reached a size of 100mm³ the animals were randomized into groups of 10 animals each. Theanimals were treated with vehicle, SP2012, SP2000, or SP2024. Thepeptides were administered subcutaneously daily. Dosing was continuedfor 3 or 4 weeks and tumor sizes were measured every 3 days usingcalipers. The tumor volume was estimated using the formula V=a²b/2 wherea and b are the smaller and larger diameters.

The tail vein assay of cancer metastasis protocol from Current Protocolsof Cell Biology was used to measure the effect of SP2024 on lungmetastasis (Elkin and Vlodaysky, 2001). 1 mL of 5×10⁵ cells/mLluciferase-transfected MDA-MB-231-luc-D3H2LN (Caliper Biosciences) cellsuspension was slowly injected into nude mice (nude mice are easier toimage because of their lack of fur). Two hours before cell inoculationvehicle or 10 mg/kg SP2024 were administered subcutaneously on a dailybasis. Bioluminescence to detect metastasis was measured by IVIS imaging(Caliper Biosciences) via IP injection of 150 mg/kg D-luciferin.

MRI of vascular volume and permeability surface-area product (PSP) weredetermined as follows. Mice with volume-matched tumors were treated withSP2024 or vehicle and imaged 24 h post treatment. The mice wereanesthetized and the tail vein was catheterized before placing theanimal in the spectrometer. All imaging studies were performed on a 4.7T Bruker Avance spectrometer, as previously described (Raman et al.,2006; Bhujwalla et al., 2001) using a home-built solenoid coil placedaround the tumor. Briefly, multislice relaxation rate (1/T1) maps wereobtained by a saturation recovery method combined with fast T1SNAPSHOT-FLASH imaging (flip angle of 10°, echo time of 2 ms). First, anMo map with a recovery delay of 7 s was acquired following which imagesof 4 slices (1 mm thick), acquired with an in-plane spatial resolutionof 125 mm (128×128 matrix, 16 mm field of view, 8 averages), wereobtained for three relaxation delays (100 ms, 500 ms and 1 s). These T1recovery maps were obtained before i.v. administration of 0.2 ml of 60mg/mL albumin-GdDTPA in saline (dose of 500 mg/kg) and repeated sixtimes starting 3 min after i.v. injection of albumin-GdDTPA.Albumin-GdDTPA was synthesized based on the method of Ogan et al.(1988). Relaxation maps were reconstructed from data sets for threedifferent relaxation times and the Mo dataset on a pixel-by-pixel basis.At the end of the imaging studies, the animals were sacrificed, 0.5 mlof blood was withdrawn from the inferior vena cava or tail vein, the T1of blood was measured, and tumors were excised and fixed in 10% bufferedformalin for sectioning and staining. Vascular volume and PSP maps weregenerated from the ratio of D(1/T1) values in the images to that ofblood. The slope of D(1/T1) ratios versus time in each pixel was used tocompute PSP, and the intercept of the line at zero time was used tocompute vascular volume, corrected for permeability of the vessels. Thedetectable areas of vascular volume and permeability as well as absolutevalues of vascular volume and permeability were analyzed.Three-dimensional volume data were processed with anoperator-independent computer program that enabled selection, mappingand display of the regions. Volume fractions of the regions weredetermined using the histogram analysis of the volume data. Values ofvascular volume and PSP were computed for every voxel in the tumor witha routine written using Interactive Data Language (IDL, ResearchSystems, Boulder, Colo.). Statistical significance (p<0.05) within eachexperiment using the independent replicates was assessed using standardstatistical assessments such as Student's t-test and ANOVA accompaniedby Dunnett's test if different sets of data were compared to one group.

The original peptide, SP2000 (SEQ ID No:19), was substituted at the twoCysteine positions to determine what effect the elimination of theCysteine residues would have on activity (Table 2). FIG. 1 shows theresults of a proliferation assay. All of the peptides with the Cysteinessubstituted inhibited the proliferation of HUVECs with the most potentactivity shown by SP2024 (SEQ ID No:11) (IC₅₀ value of 1.29 μM).

The inhibition activity of the substituted peptides in adhesion ofHUVECs also was assessed and the results are shown in FIG. 2. All of thepeptides showed inhibition of adhesion when compared to the control.

Migration assays were consistent with the adhesion assays anddemonstrated inhibition of migration of HUVECs for all the peptides whencompared to the control at a peptide concentration of 50 μM (FIG. 3).

FIG. 4 illustrates inhibition of migration with peptide SP2012 (SEQ IDNo:3). Panel A shows a wound healing assay with the migration of cellsin the presence or absence of peptide under constant concentration ofchemoattractant (complete media). The peptide concentration was variedat 0.5 μM, 5 μM, and 50 μM. Migration of MDA-MB-231 cells into therestricted area was quantified after 17 hours of incubation (Panel B).The 50 μM and 5 μM treatments were statistically significant versus thecontrol (p<0.05). Error bars depict SEM.

FIG. 5 illustrates another experiment showing the inhibition of activityin adhesion at 50 μM (Panel A) and the inhibition of activity inmigration at 50 μM (Panel B) and 5 μM (Panel C). The results again showthat all the peptides exhibit inhibition of adhesion and migration ofHUVECs. The inhibitory activity in the adhesion assay was quite similarto that of the parent compound (SP2012; SEQ ID No:3), resulting inalmost complete inhibition at 50 μM. However, the activity in migrationwas significantly increased from 60% in the parent compound (SP2012; SEQID No:3) to >90% in the modified peptides. Thus the activity wasinvestigated at 5 μM, a much lower concentration, and it was observedthat SP2022 (SEQ ID No:9) showed similar activity to the parent compound(SP2012; SEQ ID No:3) while the other compounds SP2023 through SP2027(SEQ ID Nos:10-14) showed much greater activity. The * indicatesstatistical difference (p<0.05) in activity in comparison to SP2012. They-axis range is reduced to 0-20% for Panel A and 0-50% for Panel B.

The peptides with the substituted cysteines were further tested usingthe tube formation assay (FIG. 6A and FIG. 6B). The control contained15,000 HUVECs/well plated in complete media. Each well contained peptideplated in complete media all plated on matrigel (50 μL/well). Theresults showed that all of the Cysteine-substituted peptides hadinhibitory activity on tube formation.

TABLE 2 Summary of Cysteine Substitutions in the Full Peptide Tube MDA-Peptide Proliferation Migration Adhesion formation MB-231 No./SEQInhibition Inhibition Inhibition Inhibition xenograft ID No.Peptide sequence (IC50 in uM) (50 uM) (100 uM) (100 uM) inhibitionSP2000/ LRRFSTMPFMFCNINN 15.5     80%    95% Complete Active 19 VCNSP2012/ LRRFSTMPFMFAbuNI 22      68% 93.14% Complete Active 3 NNVAbuN(50 uM) (10 uM)     0% (5 uM) SP2002/ LRRFSTMPFMFGNINN 31.4   87.9%Complete 2 VGNF (50 uM) SP2022/ LRRFSTMPFMFSNINN 16.9   8.95% 96.49% 9VSNF  (5 uM) SP2023/ LRRFSTMPFMFANINN  2.47 31.18% 96.29% Complete VANF(5 uM) SP2024/ LRRFSTMPFMFININN  1.29 37.73% 94.45% Complete Active 11VINF  (5 uM) SP2025/ LRRFSTMPFMFTNINN 13.5  18.19% 96.48% Complete 12VTNF  (5 uM) SP2026/ LRRFSTMPFMF  8.23 11.43% 96.61% Complete 13(AllyGly)NINNV  (5 uM) (AllyGly)NF SP2027/ LRRFSTMPFMFVNINN  6.04 27.02%96.75% Complete 14 VVNF  (5 uM)

Example 2 Alanine and Other Substitutions in the Full Peptide

With the peptide SP2012 (SEQ ID No:3) as a reference, Alanine was usedto substitute for other amino acids in the SP2012 (SEQ ID No:3) sequence(Table 3). These peptides all showed inhibition of proliferation (FIG.7) and inhibition of adhesion at 50 μM of HUVECs (FIG. 8). Theinhibition of migration at 50 μM was significantly greater for thepeptides with Alanine substitutions compared to SP2012 (FIG. 9). One ofthe peptides, SP2015 (SEQ ID No:4), was tested in the HUVEC tubeformation assay (FIG. 10). SP2015 (SEQ ID No:4) showed significantinhibition of tube formation.

TABLE 3 Summary of Alanine Substitutions in the Full Peptide PeptideTube No./ Proliferation Migration Adhesion formation SEQ ID InhibitionInhibition Inhibition Inhibition No. Peptide sequence (IC50 in uM)(50 uM) (100 uM) (100 uM) SP2015/ LRRFSTMPAMF(Abu)NI 16.8   100%    96%Complete 4 NNV(Abu)NF 19.48 SP2016/ LRRFSTMPFAF(Abu)NI 11.3  99.6%   96% 5 NNV(Abu)NF  2.82 SP2017/ LRRFSTMPFMA(Abu)NI  9.63  100% 95.5%%6 NNV(Abu)NF 32.9  SP2018/ LRRFSTMPF(Nle)F 18.2   100% 90.9%% 7(Abu)NINNV(Abu)NF  9.14 SP2019/ LRRFSTMPFM(4- 15.9    98%  95.9% 8ClPhe)(Abu)NINNV 18.5  (Abu)NF

Example 3 C-Terminal Deletions

Tables 4 and 5 list the family of peptides that were generated byeliminating residues from the C-terminus of the parent peptide (SP2012;SEQ ID No:3). Truncations that eliminated the NINNV (SEQ ID No:36)sequence showed loss of inhibitory activity in proliferation ofendothelial cells. Peptides that include representative residues fromthis critical sequence (i.e. SP2028 (SEQ ID No:20), SP2029 (SEQ IDNo:21), SP2032 (SEQ ID No:22) and SP2033 (SEQ ID No:23)) demonstratedpotent activity in inhibiting proliferation, even improved activity ascompared to the parent compound. The substitution of Asparagine, ahydrophilic residue, by Valine, a hydrophobic amino acid led to loss inactivity (SP2028 (SEQ ID No:20) vs. SP2006 (SEQ ID No: 18)). However, ifin addition the Abu residue was replaced by Threonine, the new compoundregained activity (SP2029 (SEQ ID No:21) vs. SP2006 (SEQ ID No:18)).Substitution of Abu in SP2028 (SEQ ID No:20) by a Threonine (SP2032; SEQID No:22), resulted in a reduction in activity while its replacement byIsoleucine maintained the same level of activity (SP2033; SEQ ID No. 23)thus confirming that the nature of the amino acid residue at thisposition is important for the inhibition of proliferation.

FIG. 11 shows the ability of the C-terminal deletions peptides toinhibit adhesion and migration of endothelial cells. Panel A lists theamino acid sequences of the peptides used in this series of experiments.Panel B illustrates an adhesion experiment at 100 μM peptideconcentration in which significant inhibition can be seen for peptideSP2006 (SEQ ID No:18). Panel C shows a migration experiment at a peptideconcentration of 50 μM. Again, inhibition can be seen for SP2006 (SEQ IDNo:18). Panel D shows the inhibition of adhesion for SP2006 (SEQ IDNo:18) at a concentration of 50 μM.

FIG. 12 again illustrates the inhibitory activity of peptides generatedby deletions from the C-terminus of SP2012 in HUVEC adhesion (Panel A)and migration assays (Panel B). The same four peptides that exhibitedactivity in the inhibition of proliferation were also very potent ininhibiting adhesion and migration, but did not display increasedactivity over the parent compound SP2012 (SEQ ID No:3). In theproliferation assay they were more potent than SP2012 (SEQ ID No:3). Inaddition SP2006 (SEQ ID No: 18), which did not inhibit proliferation,exhibited inhibitory activity in both the adhesion and migration assays.The significance of the NINNV (SEQ ID No:36) sequence is supported bythe absence of activity in the fragments which lack this region (SP2007(SEQ ID No:24), SP2008 (SEQ ID No:25), SP2013 (SEQ ID No:26) and SP2014(SEQ ID No:27)). These results also are in accordance with theinhibitory activity in the proliferation assay.

FIG. 13 shows the HUVEC tube formation assay for the C-terminal deletionpeptides. Images shown are compared to an untreated control in completemedia. Inhibition of tube formation was seen for SP2006 (SEQ ID No:18),SP2028 (SEQ ID No:20), SP2029 (SEQ ID No:21), SP2032 (SEQ ID No:22), andSP2033 (SEQ ID No:23).

TABLE 4 Summary of C-terminal Deletions Peptide Tube No./ ProliferationMigration Adhesion formation SEQ ID Inhibition Inhibition InhibitionInhibition No. Peptide sequence (IC50 in uM) (50 uM) (100 uM) (100 uM)SP2004/ LRRFSTMPFMF-OH Inactive Inactive 28 SP2006/ LRRFSTMPFMFAbuNINV56.2     17%   95%% Complete 18 -OH SP2007/ LRRFSTMPFMFAbu InactiveInactive    11% Inactive 24 SP2008/ LRRFSTMP Inactive Inactive  -0.1%Inactive 25 SP2013/ LNRFSTMPF-OH Inactive Inactive   0.3% Inactive 26SP2014/ LRRFSTNlePFNleF-OH Inactive Inactive   2.9% Inactive 27 SP2028/LRRFSTMPFMFAbuNINN  9.45 48.87%  96.8% Complete 20 SP2029/LRRFSTMPFMFTNINV 31.09 54.13% 93.75% Complete 21 SP2032/LRRFSTMPFMFTNINN 16.91 32.45%    63% Complete 22 SP2033/LRRFSTMPFMFININN 15.06 42.01%    94% Complete 23

TABLE 5 C-terminus Truncations and Peptide Proliferation ActivityPeptide Number Modification IC₅₀ ± No./SEQ Peptide of from the 95% CIID No. Name Peptide sequence residues SP2012 (μM) SP2012/3 SP2012LRRFSTMPFMFAbuNI 20 Parent 48.1 ± 23.1 NNVAbuNF peptide for this studySP2004/28 SP2004 LRRFSTMPFMF 11 Truncated 9 >100 residues SP2006/18SP2006 LRRFSTMPFMFAbuNI 16 Truncated 4 >100 NV residues SP2007/24 SP2007LRRFSTMPFMFAbu 12 Truncated 8 >100 residues SP2008/25 SP2008 LRRFSTMP  8Truncated 12 >100 residues SP2013/26 SP2013 LNRFSTMPF  9Truncated 12 >100 residues & substituted one Arginine with AsparigineSP2014/27 SP2014 LRRFSTXPFXF- 11 Truncated 9 >100 X = NorLeu residues &substituted Methionine with NorLeucine SP2028/20 SP2028 LRRFSTMPFMFAbu16 Truncated 4 5.1 ± 2.7 NINN residues SP2029/21 SP2029 LRRFSTMPFMFTNI16 Truncated 4 4.8 ± 2.8 NV residues & substituted the Abu residues withThreonine SP2032/22 SP2032 LRRFSTMPFMFTNI Truncated 4 10.3 ± 4.1  NNresidues & substituted the Abu residues with Threonine SP2033/23 SP2033LRRFSTMPFMFINI 16 Truncated 4 3.8 ± 1.7 NN residues & substitutedthe Abu residues with Isolucine

Example 4 N-Terminal Deletions

The peptides generated by removing amino acids from the N-terminus arelisted in Table 6. In contrast with the previous series of C-terminustruncations the peptides that lack residues from the N-terminus arevirtually inactive at inhibiting proliferation of HUVEC.

FIG. 14 illustrates the inhibitory activity of compounds generated bytruncations from the N-terminus in adhesion (Panel A) and migration(Panel B) of HUVECs. These compounds displayed a range of inhibitoryactivity in the adhesion and migration assays. For the purpose ofcomparison, any compound displaying an inhibitory potential of greaterthan 30% (in the graphs a % inhibition from control lower than 70%) wasconsidered active. Thus in the group of truncations from the N-terminus,peptides SP2010 (SEQ ID No:29) and SP2011 (SEQ ID No:30) showed lowinhibitory activity in the adhesion assay and SP2009 (SEQ ID No:31)showed inhibitory activity in the migration assay. All these compoundsincluded the critical NINNV fragment. The peptides active in adhesioninhibition contained residues which extended in both directions from theNINNV (SEQ ID No:36) sequence while SP2009 (SEQ ID No:31) which onlyinhibited migration included additional residues to the C-terminus ofthe NINNV sequence. Thus the context in which the NINNV (SEQ ID No:36)region is presented seems to play a role in influencing the type ofactivity that a compound will demonstrate.

TABLE 6 Summary of N-terminal Deletions Peptide Tube MDA- No./Proliferation Migration Adhesion formation MB-231 SEQ ID InhibitionInhibition Inhibition Inhibition xenograft No. Peptide sequence(IC50 in uM) (50 uM) (100 uM) (100 uM) inhibition SP2009/ NINNVAbuNF-OHInactive 55%     0% Partial 31 SP2010/ FIVIFAbuNINNVAbu 155   13% 82.69%Complete 29 NF-OH SP2011/ STMPFMFAbuNINN  51.4 Inactive     0% PartialActive 30 VAbuNF-OH

Example 5 Deletions from Both the N- and C-Terminal Ends

Peptide fragments generated by removing residues from both termini arelisted in Table 7. The proliferation, adhesion, and migration inhibitoryactivity suggests that shorter fragments were less active even if theyretained most of the NINNY (SEQ ID No:36) sequence (SP2021 (SEQ IDNo:32) and SP2030 (SEQ ID No:33). However, addition of two more residuesto the N-terminus of the NINNV (SEQ ID No:36) fragment along with thereplacement of Abu by Threonine (SP2031; SEQ ID No:34) restored activityto similar levels as the larger 16 amino acid C-terminus truncations.These results in conjunction with the fact that splitting the moleculein two almost equal pieces led to an inactive and active compound,SP2004 (SEQ ID No:28) and SP2020 (SEQ ID No:35) respectively, indicatesthat the presence of particular residues is crucial. These resultssuggest that the NINNV (SEQ ID No:36) sequence is critical to theinhibitory activity of our peptides; however, additional amino acidresidues on either end (SP2031; SEQ ID No:34) or just at the C-terminusimprove activity.

The pattern of activity in adhesion and migration inhibition by some ofthe fragments generated by truncations from both termini and from theN-terminal deletions is presented in FIG. 15. Panel A lists the aminoacid sequences of the peptides used in this series of experiments. PanelB illustrates the adhesion assays, Panel C shows the migration assays,and Panel D illustrates the proliferation assays. Table 7 lists asummary of the results of each of these experiments.

Another experiment was performed with only the peptides truncated atboth ends compared to SP2012 (SEQ ID No: 3) (FIG. 16). The SAR wassimilar to the one observed in inhibitory activity of proliferation; theabsence of the NINNV (SEQ ID No:36) sequence led to inactivation ofthese compounds (SP2021 (SEQ ID No:32) and SP2030 (SEQ ID No:33)).SP2031 (SEQ ID No:34) which includes the NINNV (SEQ ID No:36) sequencealong with substitution of Threonine in lieu of the Abu residuedisplayed robust activity across all assays; proliferation inhibition(IC₅₀ 4.6 μM), adhesion (>80 inhibition), and migration (>50%inhibition).

Some of the peptides with N-terminal deletions and both N-terminal andC-terminal deletions were tested in the tube formation assay (FIG. 17).SP2010 (SEQ ID No:29) and SP2020 (SEQ ID No:35) at 100 μM showedcomplete inhibition of tube formation.

TABLE 7 Summary of Deletions from both the N- and C-terminal Ends TubePeptide Proliferation Migration Adhesion formation No./SEQ InhibitionInhibition Inhibition Inhibition ID No. Peptide sequence (IC50 in uM)(50 uM) (100 uM) (100 uM) SP2020/ F(Abu)NINNV(Abu)N Inactive 31.89%84.4% Complete 35 SP2021/ F(Abu)NIN Inactive Inactive    0% Inactive 32SP2030/ FAbuNINV 144.5  -9.5%    0% Inactive 33 (-1.35%) SP2031/FTNINNVTN 214   10.21%   85% Active 34

TABLE 8 Truncations from both Termini and Peptide Proliferation ActivityPeptide Number Modification IC₅₀ ± No./SEQ Peptide of from the 95% CIID No. Name Peptide sequence residues SP2012 (μM) SP2012/3 SP2012LRRFSTMPFMFAbuNI 20 Parent 48.1 ± 23.1 NNVAbuNF peptide for this studySP2020/35 SP2020 FAbuNINNVAbuN  9 Truncated 11 9.8 ± 4.6 residuesSP2021/32 SP2021 FAbuNIN  5 Truncated 15 >100 residues SP2030/33 SP2030FAbuNINV  6 Truncated 14 >100 residues SP2031/34 SP2031 FTNINNVTN  9Truncated 11 4.7 ± 1.5 residues & substituted the Abu residues withThreonine

Example 6 Substitutions and Second Generation Substitutions in the FullLength Peptide

The truncation studies demonstrated that the Abu positions (in SP2012;SEQ ID No:3) are important in maintaining strong activity in theinhibition of proliferation of endothelial cells thus additionalsubstitutions were introduced at these positions (12 and 18). Thesesubstitutions are presented in Table 9. The inhibitory activity of thesepeptides was not affected when the Abu was substituted by Serine(SP2022; SEQ ID No:9), Threonine (SP2025; SEQ ID No:12), AllyGly(SP2026; SEQ ID No:13) or Valine (SP2027; SEQ ID No:14) thushydrophilicity at these locations is not crucial. Hydrophobic residuessuch as Alanine or Isoleucine increased the activity dramatically, 2.8and 6.5 times, respectively.

One of the most active full length peptides, SP2024 (SEQ ID No:11),which contains Isoleucines instead of Abu residues present in the parentpeptide (SP2012; SEQ ID No:3) was modified to generate additional 2^(nd)generation peptides. These compounds are illustrated in Table 10. Two ofthese peptides (SP2034 (SEQ ID No:15) and SP2035 (SEQ ID No:16)demonstrated submicromolar activity in proliferation (IC₅₀ 0.54±0.19 and0.94±0.38 μM respectively), a remarkable increase in activity from theparent peptide, SP2012 (SEQ ID No:3) (IC₅₀ 48.1±23.1 μM). It isinteresting to note that substitution of the second Methionine inposition 10 (SP2034; SEQ ID No:15) with the non-natural amino acidd-Alanine or Alanine led to considerable increases in activity, whilethe additional substitution of the Methionine at position 7 resulted ina 10-fold loss of activity compared to substitution of Methionine atposition 10 (SP2036; SEQ ID No:17).

The introduction of methionine substitutions led to significantincreases in activity in adhesion inhibition as illustrated in FIGS. 18and 19. The parent compound (SP2012; SEQ ID No:3) exhibited an IC₅₀ inadhesion inhibition of 2.39±1.55 μM, which was not affected by theintroduction of the Isoleucine in lieu of the Abu residues at positions12 and 18 (SP2024 (SEQ ID No:11); IC₅₀ 2.35±1.3 μM). The IC₅₀ was notaffected by further substitutions of either one Methionine (position 10)or both (position 7 and 10) with Alanine (SP2035 (SEQ ID No:16); IC₅₀2.51±0.88 μM and SP2036 (SEQ ID No:17); IC₅₀ 2.75±1.22 μM). However,this activity was increased four times by the introduction of D-Alaninein lieu of the Methionine at position 7 (SP2034 (SEQ ID No:15); IC₅₀0.55±0.37 μM). Similarly the inhibition of proliferation was increasedfrom the parent SP2012 (SEQ ID No:3; IC₅₀ 48.1±23.1 μM) by one order ofmagnitude in SP2024 (SEQ ID No:11; IC₅₀ 7.45±5.7 μM) and even further bytwo order of magnitudes from SP2012 (SEQ ID No:3) to SP2034 (SEQ IDNo:15) and SP2035 (SEQ ID No:16) with IC₅₀ 0.54±0.19 μM and IC₅₀0.98±0.38 μM respectively.

Since the ability to inhibit tube formation is a comprehensiveassessment of the anti-angiogenic potential, the activity of thesepeptides on HUVEC tube formation was determined (FIG. 20). Theinhibition of tube formation was tested at a high dose, 100 μM, so thatthe inhibition was obvious thus eliminating the need for quantification.Based on the results from adhesion and migration it was expected that inthe group of C-terminus truncation peptides SP2028 (SEQ ID No:20),SP2029 (SEQ ID No:21), SP2032 (SEQ ID No:22) and SP2033 (SEQ ID No:23)would have activity in the inhibition of tube formation. Surprisingly,SP2006 (SEQ ID No:18) also was active at inhibiting tube formation, eventhough it was inactive in the migration assay. The compound had 18%activity in inhibiting migration which was below the threshold of atleast 70% activity. However SP2006 (SEQ ID No:18) had strong activity inthe adhesion assay thus it supports its activity in inhibition of tubeformation. On the other hand the N-terminus truncated SP2011 (SEQ IDNo:30) showed no activity in tube formation as expected because it wasinactive in the migration assay and it only had minimal activity inadhesion. Both SP2009 (SEQ ID No:31) and SP2010 (SEQ ID No:29) displayedactivity in inhibiting tube formation as expected and were active atinhibiting adhesion or migration, respectively.

The group of peptides generated by truncations from both ends includedtwo active compounds (SP2020 (SEQ ID No:35) and SP2031 (SEQ ID No:34))and two inactive compounds (SP2021 (SEQ ID No:32) and SP2031 (SEQ IDNo:34)). All compounds in the substitution and 2^(nd) generationsubstitution group were very active at inhibiting tube formation whichis expected as their potency in migration and adhesion was eitherincreased or not affected by the substitutions.

TABLE 9 Amino Acid Substitutions and Peptide Activity in ProliferationPeptide Modification No./SEQ Peptide from IC₅₀ ± 95% ID No. NamePeptide sequence the SP2012 CI (μM) SP2012/3 SP2012LRRFSTMPFMFAbuNINNVAbuNF Parent peptide for 48.1 ± 23.1 this studySP2022/9 SP2022 LRRFSTMPFMF S NINNV S NF Abu residues 44.9 ± 29.4replaced by Serine SP2023/10 SP2023 LRRFSTMPFMF A NINNV A NFAbu residues 17.3 ± 6.0  replaced by Alanine SP2024/11 SP2024LRRFSTMPFMF I NINNV I NF Abu residues 7.45 ± 5.7  replaced by IsoleucineSP2025/12 SP2025 LRRFSTMPFMF T NINNV T NF Abu residues 63.8 ± 35.0replaced by Threonine SP2026/13 SP2026 LRRFSTMPFMF(AllyGly)NINNVAbu residues 47.3 ± 29.2 (AllyGly)NF replaced by AllyGly SP2027/14SP2027 LRRFSTMPFMF V NINNV V NF Abu residues 33.9 ± 17.9replaced by Valine

TABLE 10 Inhibition of HUVEC Proliferationby SP2024, SP2034, SP2035, and SP2036 Peptide Modification IC₅₀ ±No./SEQ Peptide from the original 95% CI ID No. Name Peptide sequencesequence SP2000 (μM) CI SP2024/11 SP 2024 LRRFSTMPFM FI Cysteines 7.45 ±5.7  NINNVI NF replaced by Isolucine SP2034/15 SP 2034 LRRFSTMPFdA FISecond Methionine 0.54 ± 0.19 NINNVI NF replaced by dAlanine 5P2035/16SP 2035 LRRFSTMPFA FI Second Methionine 0.94 ± 0.38 NINNVI NF replacedby Alanine SP2036/17 SP 2036 LRRFSTAPFA FI Both Methionine 9.97 ± 8.3 NINNVI NF replaced by Alanine

TABLE 11 Summary of Activity for All Compounds Activity Peptide inActivity Activity Activity No./SEQ Peptide Prolif- in in in Tube ID No.Name Peptide sequence eration Adhesion Migration formation SP2024/11SP2012 LRRFSTMPFMFAbuNINNVAbuNF Yes Yes Yes Yes SP2004/28 SP2004LRRFSTMPFMF No No No No SP2006/18 SP2006 LRRFSTMPFMFAbuNINV No Yes NoYes SP2007/24 SP2007 LRRFSTMPFMFAbu No No No No SP2008/25 SP2008LRRFSTMP No No No No SP2013/26 SP2013 LNRFSTMPF No No No No SP2014/27SP2014 LRRFSTXPFXF- X = NorLeu No No No No SP2028/20 SP2028LRRFSTMPFMFAbuNINN Yes Yes Yes Yes SP2029/21 SP2029 LRRFSTMPFMFTNINV YesYes Yes Yes SP2032/22 SP2032 LRRFSTMPFMFTNINN Yes Yes Yes Yes SP2033/23SP2033 LRRFSTMPFMFININN Yes Yes Yes Yes SP2009/31 SP2009 NINNVAbuNF NoNo Yes Yes SP2010/29 SP2010 FMFAbuNINNVAbuNF No Yes No Yes SP2011/30SP2011 STMPFAbuNINNVAbuNF No Yes No No SP2020/35 SP2020 FAbuNINNVAbuNYes Yes Yes Yes SP2021/32 SP2021 FAbuNIN No No No No SP2030/33 SP2030FAbuNINV No No No No SP2031/34 SP2031 FTNINNVTN Yes Yes Yes Yes SP2022/9SP2022 LRRFSTMPFMF S NINNV S NF Yes Yes Yes Yes SP2023/10 SP2023LRRFSTMPFMF A NINNV A NF Yes Yes Yes Yes SP2024/11 SP2024LRRFSTMPFMF I NINNV I NF Yes Yes Yes Yes SP2025/12 SP2025LRRFSTMPFMF T NINNV T NF Yes Yes Yes Yes SP2026/13 SP2026LRRFSTMPFMF(AllyGly)NINNV Yes Yes Yes Yes (AllyGly)NF SP2027/14 SP2027LRRFSTMPFMF V NINNV V NF Yes Yes Yes Yes SP2034/15 SP2034LRRFSTMPFdA FI NINNVINF Yes Yes Yes Yes SP2035/16 SP2035LRRFSTMPFA FI NINNVINF Yes Yes Yes Yes SP2036/17 SP2036LRRFSTAPFA FI NINNVINF Yes Yes Yes Yes

The structure-activity relationship of anti-angiogenic peptides whoseoriginal peptide (SP2000; SEQ ID No:19) was derived from the α5 fibrilof type IV collagen was analyzed (Summary in Table 11). The potentcysteine-free peptide (SP2012; SEQ ID No:3), the parent peptide for thisstudy, was tested in vitro and in vivo xenograft mouse model of triplenegative breast cancer, a form of cancer which is not amendable toconventional chemotherapeutic and hormonal treatments (Avraamides, etal., 2008).

The presently disclosed subject matter provides a small family ofpeptides which were screened in a series of in vitro assays:proliferation, adhesion, migration and tube formation with macrovascularendothelial cells (HUVEC) commonly used in angiogenesis studies.Selected peptides also were tested using microvascular endothelial cells(MEC), lymphatic endothelial cells (LEC) and MDA-MB-231 breast cancercells and showed similarity of responses for these cell types. Theproliferation inhibition activity was retained when most of the sequencewas preserved (SP2028; SEQ ID No:20) or when specific substitutions wereintroduced. Shorter fragments that still maintained activity includedthe NINNV (SEQ ID No:36) sequence which was coupled with additionalamino acids on both sides for optimal activity (SP2009 (SEQ ID No:31)vs. SP2020 (SEQ ID No:35)). The in vitro activity was improved by twoorders of magnitude in comparison to that of the parent peptide, whenthe length was maintained and certain substitutions were introduced(SP2034; SEQ ID No:15). Overall adhesion and migration profiles weresimilar; generally a compound active at inhibiting adhesion would alsobe active at inhibiting migration. Similarly the activity in theinhibition of adhesion and migration was maintained if the NINNV (SEQ IDNo:36) sequence was conserved (SP2004 (SEQ ID No:28) vs. SP2028 (SEQ IDNo:20)). However, the addition of amino acids to this sequence seemed toplay a different role: if the NINNV (SEQ ID No:36) region was flankedwith residues from the C-terminus (SP2009; SEQ ID No:31) the compoundwas active in migration and not adhesion while if it was flanked withresidues from the N-terminus (SP2006; SEQ ID No:18) the activity wasreversed, i.e. the compound was active in adhesion but not migration.Despite the similarities in the activity profiles of adhesion andmigration there were a few exceptions, e.g. SP2006 (SEQ ID No:18),SP2010 (SEQ ID No:29), SP2011 (SEQ ID No:30) compounds which are activein adhesion but not in migration and compound SP2009 (SEQ ID No:31)which was active in migration but not adhesion.

Inhibition of tube formation correlates very well with activity ineither inhibition of adhesion or migration; if a compound was active ineither of those two assays it consistently demonstrated activity in theinhibition of tube formation. High concentrations of peptides were usedto assess the activity in this assay so that the inhibition was completeand there was no need for quantification of the effects. Also, twodifferent phenotypes were observed in the inhibition by these compounds;one in which the cells were clumped (e.g., FIG. 23, panel B), and theother in which the cells were less clumped and formed fragile and shorttubes (e.g., FIG. 23, panel A). The clumped profile was observed withall the compounds that inhibited adhesion while the short tubes profilewas associated with compounds which were active in migration. In thecompounds that were active in both, adhesion and migration, the clumpprofile predominated however. Live-dead staining of cells treated withthe compounds (results not shown) have indicated that cells which wereat the bottom of the clump were dead while cells which were in contactwith other cells were still alive but could not rearrange due to theirinability to migrate.

In summary, a structure-activity relationship study has been presentedherein for a family of anti-angiogenic peptides. A number of amino acidsand amino acid sequences which were important for in vitro activity ofthese compounds has been found. A short sequence, NINNV (SEQ ID No:36),was found to be important in the activity of this peptide in inhibitingproliferation, adhesion, migration and tube formation in HUVEC. Theresults were qualitatively similar to those in MEC and LEC. These appearto be the only known peptides that exhibit anti-lymphangiogenicactivity. Through specific and directed substitutions, compounds werecreated which exhibited a significant (in some cases, two orders ofmagnitude) increase in activity over the activity of parent peptide.

Example 7 Inhibitory Activity of Peptides on Multiple Cell Types

Even though HUVEC remain the most common cells used in angiogenesisassays, they are derived from veins, and thus there are questionswhether they are the most appropriate model for studying angiogenesis.It has been shown that many of the in vitro assays with HUVEC andmicrovascular endothelial cells (MEC) exhibit qualitatively similarbehaviors (Avraamides, et al., 2008). FIG. 21 shows the inhibitionactivity of the parent peptide SP2012 (SEQ ID No:3) and SP2024 (SEQ IDNo:11) in an adhesion assay (Panel A) and in a migration assay (Panel C)on lymphatic endothelial cells (LEC). This figure also shows theinhibition activity of the parent peptide SP2012 (SEQ ID No:3) andSP2024 (SEQ ID No:11) in an adhesion assay (Panel B) and in a migrationassay (Panel D) on microvascular cells (MEC) and migration inhibition onbreast cancer cells MDA-MB-231 (Panel E). These results demonstrate thatthese peptides are quite active at inhibiting adhesion and migration ofMEC. Further, the activity of these two peptides at inhibiting theadhesion and migration of lymphatic endothelial cells (LEC) and theMDA-MB-231 breast cancer cells also was strong. Thus these peptidespossess inhibitory activity towards multiple cell types thus potentiallyinhibiting the growth of blood and lymphatic vessels and also tumorgrowth and metastasis.

FIG. 22 again demonstrates the inhibitory activity of SP2024 (SEQ IDNo:11) on breast cancer cells MDA-MB-231. Panel A shows inhibition ofcell adhesion by the SP2024 (SEQ ID No:11) peptide and Panel Bdemonstrates inhibition of cell migration.

Example 8 Inhibitory Activity of Peptides In Vivo

For the tumor xenograft assay, animals were housed and treated accordingto the approved animal protocol of the Institutional Care and UseCommittee at Johns Hopkins Medical Institution (JHMI). Orthotopic breasttumors were initiated in SCID mice using MDA-MB-231 cells. 2×10⁶ cellsper 100 μL aliquot of single cell suspension were injected in the breastmannary fat pad. Tumors reached volumes of 75 mm³ to 100 mm³ inapproximately 14-21 days. Mice were randomized and arranged in groups (8mice per group) with similar tumor volumes (no statistical differenceamong averages) and treatment was commenced. Peptides were administeredonce per day intraperitoneally (i.p.) at doses of 10 mg/kg (subcutaneousdelivery was also tested with certain peptides). Paclitaxel wasadministered at 5 mg/kg once a week, and to maintain the daily treatmentregime PBS was administered on the non-treatment days. Tumors weremeasured every fourth day using calipers and the tumor volume wascalculated using the formula V=ab²/2, where a is the larger diameter andb is the smaller diameter.

A Matrigel plug assay was performed to evaluate the inhibitory effect ofthe peptides in angiogenesis and lymphangiogenesis in vivo. BasementMembrane Matrix (Matrigel, growth factor reduced, high concentration,LDEV-free, BD Biosciences) containing VEGFA (380 ng/mL) and heparin (10unit) was mixed with or without the peptides making total volume of 400μL. The final concentration of the peptides was 25 μM. DMSO contentswere controlled identically within all experimental groups. The Matrigelmixtures were subcutaneously injected on both flanks on the abdominalside of athymic nude mice under anesthesia (25% xylazine+25%acepromazine (vol/vol) in PBS giving a concentration of 50 mg/kg ofketamine and 5 mg/kg of acepromazine). Animals were housed and treatedaccording to the approved animal protocol of the Institutional Care andUse Committee at Johns Hopkins Medical Institution (JHMI). After 10 daysmice were euthanized and the gels were removed and weighed. At the endof the study, a few mice were infused intravenously with 200 μL of FITC(fluorescein isothiocyanate)-Dextran (20 mg/mL; Santa CruzBiotechnology, Santa Cruz, Calif.) and 1 h later, Matrigel plugs weresurgically removed and placed in 10% formalin (BD Biosciences, SanDiego, Calif.) for 16 hours, then washed in PBS, homogenized, andquantified in a 96-well fluorescent plate reader. Representativesections were imaged using a Nikon microscope to show the amount ofvascular invasion into the gel. Quantification of vessels was performedby using immunohistochemistry.

The ocular laser-induced choroidal neovascularization (NV) model of wetage-related macular degeneration was used for testing. 5-6-week-oldfemale pathogen-free C57BL/6 mice were anesthetized with ketaminehydrochloride (100 mg/kg body weight) and pupils were dilated. Laserphotocoagulation (75-μm spot size, 0.1-sec duration, 120 mW) wasperformed in the 9, 12, and 3 o'clock positions of the posterior pole ofeach eye with the slit lamp delivery system of an OcuLight GL diodelaser (Index, Mountain View, Calif.) and a handheld cover slip as acontact lens to view the retina. Production of a tissue bubble by thelaser, which indicates rupture of Bruch's membrane, is an importantfactor in obtaining choroidal NV; therefore, only burns in which abubble is produced are included in the study. Immediately afterlaser-induced rupture of Bruch's membrane, mice (n=15 for each dose)were given an intravitreous injection of 1 μl of phosphate-bufferedsaline (PBS) containing 0.1 μg of peptide, 0.1 μg scrambled peptide. Atday 7 the mice that received the naked peptides were dosed again whereasthe mice that received the particles were administered PBS.Intravitreous injections were done under a dissecting microscope with aHarvard Pump Microinjection System and pulled glass micropipettes. After14 days, the mice were perfused with 1 mL of PBS containing 50 mg/mL offluorescein-labeled dextran (2×10⁶ Daltons average molecular weight;Sigma-Aldrich, St. Louis, Mo.) and choroidal flat mounts were examinedby fluorescence microscopy. Images were captured with a Nikon DigitalStill Camera DXM1200 (Nikon Instruments Inc., New York, N.Y.). Imageanalysis software (Image-Pro Plus; Media Cybernetics, Silver Spring,Md.) was used to measure the total area of choroidal NV at each rupturesite with the investigator masked with respect to treatment group.

The ocular Rho/VEGF transgenic mice model of wet age-related maculardegeneration also was used to test some of the peptides. Transgenic micein which the rhodopsin promoter drives expression of VEGF inphotoreceptors have the onset of VEGF production at P7 and developextensive NV along the outer surface of the retina by P21 (Okamoto etal., 1997; Tobe et al., 1998). Mice hemizygous for the transgene (n=15for each dose) were given an intraocular injection of 1 μl of PBScontaining 0.01, 0.1, or 1 μg of peptide or scramble peptide in one eyeat P7 and P14 and at P21 the mice were anesthetized, perfused withfluorescein-labeled dextran, and the total area of neovascularization onthe outer surface of the retina was measured on retinal flat mounts byimage analysis as above.

Some of the peptides were tested in vivo using the tumor xenograft assayin mice. FIG. 23 shows the suppression of tumor growth by peptidesSP2000 (SEQ ID No:19) and SP2012 (SEQ ID No:3) (Panel A). In thisexperiment, the control group (PBS with 10% DMSO) and the experimentalgroups (10 mg/kg of peptide to mouse weight) were tested. Measurementswere performed every fourth day. Error bars depict SEM. As can be seenfrom the graph, tumor growth was inhibited by SP2000 (SEQ ID No:19) andSP2012 (SEQ ID No:3). Panel B shows the quantification of microvasculardensity after the completion of the treatment at 21 days. All groupswere statistically different from one another (p<0.05)

FIG. 24 demonstrates the inhibition of breast cancer cells MDA-MB-231 bythe peptide SP2024 (SEQ ID No:11). Panel A shows that SP2024 (SEQ IDNo:11) inhibited tumor growth significantly over the control. Panel Bdemonstrates that SP2024 (SEQ ID No:11) reduced relative vascular volumeand Panel C showed that the peptide reduced tumor permeability-surfacearea product compared to the control as measured by MRI.

FIG. 25 demonstrates that SP2024 (SEQ ID No:11) inhibits lung metastasisin an experiment with luciferase-transfected MDA-MB-231 cells injectedinto nude mice, as measured with a Xenogen IVIS system. FIG. 26 showsthe results from the injection of SP2024 (SEQ ID No:11) (0.01, 0.1, or 1μg) into the mouse eye. Both the area of choroidal neovascularization(Panel A) and the area of retinal neovascularization (Panel B) generallydecreased as the amount of SP2024 (SEQ ID No:11) was increased.

Several other peptides, SP2034, SP2035 and SP2036 (SEQ ID Nos: 15-17),also were tested in vivo. FIG. 27 shows that the injection of SP2034,SP2035, or SP2036 (SEQ ID Nos: 15-17) (0.1 μg) into a mouse eyeinhibited laser-induced choroidal neovascularization (Panel A) and thearea of retinal neovascularization in the Rho/VEGF model (Panel B). Inthis case, 0.1 μg of SP2036 (SEQ ID No:17) administered directly intothe eye inhibited VEGF mediated angiogenesis by about 70%.

REFERENCES

All publications, patent applications, patents, and other referencesmentioned in the specification are indicative of the level of thoseskilled in the art to which the presently disclosed subject matterpertains. All publications, patent applications, patents, and otherreferences are herein incorporated by reference to the same extent as ifeach individual publication, patent application, patent, and otherreference was specifically and individually indicated to be incorporatedby reference. It will be understood that, although a number of patentapplications, patents, and other references are referred to herein, suchreference does not constitute an admission that any of these documentsforms part of the common general knowledge in the art.

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Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

1.-71. (canceled)
 72. A method for treating a cancer patient comprisingadministering a pharmaceutical composition comprising an effectiveamount of a peptide to reduce or prevent lymphangiogenesis, the peptidecomprising the amino acid sequence LRRFSTXPXXXXNINNVXNF (SEQ ID No:1),wherein X is any amino acid.
 73. The method of claim 72, wherein X atposition 7 is M, A, or G; X at position 9 is F, A, Y, or G; X atposition 10 is M, A, G, dA, or Nle; X at position 11 is F, A, Y, G, or4-ClPhe; X at position 12 and position 18 are Abu, G, S, A, V, T, I, L,or AllylGly.
 74. The method of claim 72, wherein the peptide has theamino acid sequence LRRFSTMPFMFAbuNINNVAbuNF (SEQ ID No: 3).
 75. Themethod of claim 72, wherein the peptide has the amino acid sequenceLRRFSTMPFMFANINNVANF (SEQ ID No: 10).
 76. The method of claim 72,wherein the peptide has the amino acid sequence LRRFSTMPFMFININNVINF(SEQ ID No: 11).
 77. The method of claim 72, wherein the peptide has theamino acid sequence LRRFSTAPFAFININNVINF (SEQ ID No: 17).
 78. The methodof claim 72, wherein the cancer is a primary tumor.
 79. The method ofclaim 72, wherein the cancer is breast cancer.
 80. The method of claim72, wherein the cancer is lung cancer.
 81. The method of claim 72,wherein the cancer is glioblastoma.
 82. The method of claim 72, whereinthe cancer is renal cell cancer.
 83. The method of claim 72, wherein thecancer is hepatic cell cancer.
 84. The method of claim 72, wherein thecancer is head and neck cancer.
 85. The method of claim 72, wherein thepharmaceutical composition comprises the effective amount of the peptideand a pharmaceutically acceptable carrier.
 86. The method of claim 72,wherein the pharmaceutical composition is administered locally orsystemically.
 87. The method of claim 86, wherein the pharmaceuticalcomposition is formulated for parenteral delivery.
 88. The method ofclaim 87, wherein the pharmaceutical composition is administered byintramuscular, subcutaneous, intravenous, intra-hepatic, intra-lesional,intracranial, or intraperitoneal delivery.
 89. A method for treating acancer patient comprising administering a pharmaceutical compositioncomprising an effective amount of a peptide to reduce or preventlymphangiogenesis, the peptide comprising the amino acid sequenceLRRFSTMPFMFAbuNINNVAbuNF (SEQ ID No:3) or the amino acid sequenceLRRFSTMPFMFANINNVANF (SEQ ID No:10), and wherein the cancer is breastcancer, lung cancer, glioblastoma, renal cell cancer, hepatic cellcancer, or head and neck cancer.